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The Biological Role of CIP2A and Reptin in Renal Cell Carcinoma

Author: RenJuChao
Tutor: XuZhongHua
School: Shandong University
Course: Surgery
Keywords: CIP2A Reptin renal cell carcinoma prognosis invasion
CLC: R737.11
Type: PhD thesis
Year: 2012
Downloads: 122
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Abstract


BackgroundRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, and its incidence is increasing in recent years. But its pathogenesis has not been yet fully elucidated. CIP2A (Cancerous inhibitor of protein phosphatase2A) is one kind of oncoprotein that inhibits protein phosphatase2A (PP2A). Protein phosphatase2A (PP2A) is the the eukaryotes Ser/Thr histidine protein phosphatase that can antagonize the Ser/Thr histidine protein kinase activity. Recent studies found that there was a certain correlation between the dysregulation of PP2A activity and tumorigenesis. And it is generally believed that PP2A is a potential tumor suppressor. PP2A acts on the serine62of the oncogenic transcription factor c-Myc, and the dephosphorylation contributes to c-Myc protein degradation. CIP2A inhibits PP2A activity, and thus inhibits the hydrolysis of c-Myc and thus maintains the malignant phenotype of tumor cells. CIP2A plays an important role in tumor development, progression and biological behaviors. Reptin, also called RuvB12, Tip49b, ECP51, TAP54a, TIH2and discovered by several groups in the late1990s through various approaches and in several species, belongs to the family of AAA+ATPases (ATPases Associated with various cellular Activities). Reptin is a component of the INO80and SWR1chromatin-remodeling complexes and the Tip60histone acetyltransferase complex. Reptin is involved in chromatin remodeling, transcriptional regulation, DNA damage repair and telomerase activity. As shown in previous studies, Reptin plays an important role in carcinogenesis through interacting with many binding partners, including transcription factors/coregulators (i.e., β-atenin, c-Myc, PROP1, NF-κB P50, TLE, Hint1). In this study, we investigate the biological functions of CIP2A and Reptin in RCC and their clinical significances for RCC patients. ObjectiveIn this study we detect the expression of CIP2A and Reptin in renal tissues, and analyze the relationship between CIP2A/Reptin expression and clinicopathological parameters including prognosis of the RCC patients. Through experiments in vitro, we aim to do the preliminary investigations on the biological functions of CIP2A and Reptin and the pathogenic mechanism in RCC.MethodsWe detected expression of CIP2A mRNA using real-time quantitative PCR in26renal cell carcinoma specimens (15clear cell RCC,7papillary RCC and4chromophobe RCC) and15corresponding tumour adjacent tissues and6normal renal tissues. And CIP2A protein expression was analysed in107RCC tissues,19tumour adjacent tissues and6normal renal tissues by immunohistochemical staining. And associations between clinical-pathological parameters and CIP2A immunostaining were analysed use the chi-square test analysis. Survival analysis of the patients had also been done. The univariate survival analysis was done using Kaplan-Meier curve and analysed using Log-rank test. The multivariate survival analysis was done using Cox proportional hazards regression model. In vitro, two typical RCC cell lines, A498and KRC/Y cells, were employed to exam the involvement of CIP2A in RCC using small interfering RNA (siRNA). After acquiring reliable interference effects, we detected CIP2A expression in RCC cells with different treatment using RT-PCR and Western-blot. We detected the relationship between CIP2A and RCC cell proliferation using colony formation assay. And the involvement of CIP2A in the cell migration and invasion by means of down-regulating CIP2A expression using small interfering RNA (siRNA) were examed with scratch migration assay and matrigel invasion assay.We detected expression of Reptin protein using immunohistochemical staining in81renal cell carcinoma specimens (65clear cell RCC,10papillary RCC and6chromophobe RCC) and25corresponding tumour adjacent normal renal tissues. And then associations between clinical-pathological data and Reptin immunostaining were analysed use the chi-square test. Survival analysis of the patients had also been done. The univariate survival analysis was done using Kaplan-Meier curve and analysed using Log-rank test. The multivariate survival analysis was done using Cox proportional hazards regression model. In vitro, two typical RCC cell lines, A498and KRC/Y cells, were employed to exam the involvement of Reptin in RCC using small interfering RNA (siRNA). After acquiring reliable interference effects, we detected Reptin expression in RCC cells with different treatment using RT-PCR and Western-blot. Clonogenesis, cellular senescence and cell cycle distribution were examined by colony formation assay, β-Galactosidase staining and flow cytometry, respectively. Cell migration and invasion capability were determined by scratch migration assay and Matrigel invasion assay.ResultsResults of real-time quantitative PCR show that the expression level of CIP2A mRNA in RCCs (89.58±11.81) is significantly higher than that in corresponding tumor adjacent tissues (5.27±0.56) and normal renal tissues (3.67±0.92). Especially, level of CIP2A mRNA in clear cell RCC (130.67±1.48) is significantly higher than that in papillary RCC (36.14±7.71), and in chromophobe RCC (29.00±6.06)(P<0.001). Immunohistochemisty shows that the presence of the CIP2A protein staining was found in75of107(70%) of cancer samples, whereas only6of19(32%) tumour adjacent tissues and1of6(17%) normal renal tissues exhibited weak or diffuse CIP2A expression (P<0.001). CIP2A protein is predominantly immunolocalized to cytoplasm and occasionally to nuclear, the expression levels were higher than the normal renal tissues and positively correlated with pathological tumor stage (P<0.001), histological grade (P=0.004), clinical stage (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P<0.001) and histological type (P=0.022). There is no significant correlation between CIP2A expression and patients’gender (P=0.125) and age (P=0.082). Survival analysis indicats that increased CIP2A expression correlated with poor patients’survival. Multivariate analysis shows that high-CIP2A expression is an independent prognostic factor for RCC patients, especially for clear cell RCC patients. In vitro, CIP2A mRNA and protein expression was downregulated by siRNA targeting CIP2A in A498and KRC/Y cells. Meanwhile, the level of c-Myc protein expression is in accordance with CIP2A expression in above cells. Results of colony formation assay revealed that the proliferative capacity of the two RCC cell lines was not significantly inhibited after interferring CIP2A expression. The migration capability of A498and KRC/Y transfected with CIP2A siRNA was markedly inhibited at24h (migrating distance of cell:A498control vs CIP2A siRNA:100±11vs16±2, P<0.001; KRC/Y control vs CIP2A siRNA:117±15vs16±2, P<0.001) and48h (A498control vs CIP2A siRNA:235±5vs28±3, P<0.001; KRC/Y control vs CIP2A siRNA:230±5vs47±8, P<0.001) after scratch. Matrigel invasion assay demonstrated that the number of invasive cells passing through the filter were significantly reduced after knocking-down of CIP2A in A498(count of cells invaded per field:control vs CIP2A siRNA:115±5vs24±2, P<0.001) and KRC/Y cells (control vs CIP2A siRNA:71±4vs12±3, P<0.001).Immunohistochemisty staining showed that the presence of Reptin protein staining was found in56of81(69.1%) of RCC samples, whereas only6of25(24.0%) tumour adjacent tissues exhibited weak or diffuse Reptin expression (P<0.001). Reptin protein was present both in cytoplasmic and in nuclear compartments of tumor cells, but only weak or diffuse Reptin staining was observed in some renal tubule or glomeruli in tumor adjacent renal tissues. The expression levels positively correlated with pathological tumor stage (P=0.020) and histologic grade (P=0.040), but not with gender (P=0.172), age (P=0.779), lymph node metastasis (P=0.892), distant metastasis (P=0.434), clinical stage (P=0.139) and histology type (P=0.499). Furthermore, we found that the localization of Reptin was associated with histological grade. The cytoplasmic staining of Reptin (31of56) indicated poor differentiation of the RCC patients, while only nuclear staining of Reptin protein (25of56) was related to well-differentiated tumor (P=0.001). Univariate5-year overall survival revealed that patients with an advanced pathological tumor stage (P<0.001), distant metastasis (P<0.001) and high histological grade (P=0.004) had worse survival outcomes. The overall survival (OS) rate of patients with positive Reptin expression was not significantly lower than that with negative Reptin expression (P=0.176). However, cytoplasmic localization of Reptin staining was associated with poor prognosis and only nuclear localization or without Reptin expression predicted a favorable survival outcome of RCC patients (P=0.001). In addition, the multivariate analysis indicated that cytoplasmic localization of Reptin expression was an independent prognostic factor for RCC patients (P=0.048). In vitro, targeting of Reptin mRNA expression in A498, KRC/Y cells with special siRNA, the results of colony formation assay showed impaired clonogenic potential of RCC cells. The foci numbers decreased significantly in Reptin-depleted A498and KRC/Y cells compared to those treated with control siRNA (control vs Reptin siRNA, A498:69±4vs13±2, P<0.001; KRC/Y:46±3vs3±0.5, P<0.001). The results of β-Galactosidase staining showed senescence of RCC cells induced by the depletion of Reptin. The percentage of β-Gal-positive in control RCC cells was significantly lower than that in Reptin siRNA-treated cells (percentage of SA-β-Gal-positive cells:control vs Reptin siRNA, A498:6.1±0.6vs51.8±3.8, P <0.001; KRC/Y:3.6±0.4vs36.7±1.8, P<0.001). The results of flow cytometry for cell cycle analysis showed cell-cycle arrest induced by Reptin depletion. Flow cytometry analysis demonstrated that these Reptin siRNA-treated cells underwent growth arrest at the G1phase (percentage of cell cycle in G1phase:control vs Reptin siRNA, A498:46.2±2.0vs56.8±1.3, P<0.001; KRC/Y:51.5±0.5vs66.3±0.7, P <0.001). The results of scratch migration assay showed attenuated migration capacity of RCC cells by Reptin depletion. The migration capability of A498and KRC/Y transfected with Reptin siRNA was markedly inhibited at24h (migrating distance of cell:control vs Reptin siRNA, A498:182±6vs149±9, P<0.001; KRC/Y:80±10vs12±3, P<0.001) and48h (A498:229±6vs180±8, P<0.001; KRC/Y:188±10vs24±6, P<0.001) after scratch. The results of Matrigel invasion assay showed attenuated invasion capacity of RCC cells by Reptin depletion. The number of invasive cells passing through the filter were significantly reduced after knocking-down of Reptin in A498(count of cells invaded per field:control vs Reptin siRNA:96±7vs20±2, P <0.001) and KRC/Y cells (100±4vs32±3, P<0.001).Conclusions CIP2A is over-expressed in renal cell carcinomas (RCCs) especially in clear cell RCC, and plays an important role in RCC metastasis. Higher expression of CIP2A is positively correlated with the aggressive phenotype of RCCs, and predicts a poor outcome of patients. Therefore, CIP2A may be a novel target for the prevention and treatment of RCC metastasis and recurrence. Reptin is over-expressed in renal cell carcinomas (RCCs). Cytoplasmic expression of Reptin positively correlates with the poor differentiation of RCCs, and predicts an unpleasant outcome for patients. Reptin is required for sustained proliferation and overcome of cellular senescence of malignant cells. Inhibition of Reptin expression leads to impaired clonogenic potential and induced senescence of RCC cells as well as attenuated migration and invasion ability. Therefore, Reptin may be a novel target for prevention and treatment of RCC.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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